Project Summary/Abstract Malaria infects over 200 million people and kills over 400,000 children annually. Most infections result from Plasmodium falciparum and are detected by rapid diagnostic tests (RDTs), which enable reliable clinical diagnosis in field settings. However, in multiple settings ?diagnosis-resistant? P. falciparum parasites have emerged that do not express the antigen targeted by most tests, Histidine-Rich Protein 2 (HRP2). These HRP2-deleted parasites result in false-negative RDT results, which both prevent proper treatment and undermine investments in RDT-guided malaria treatment in the clinic. For this reason, there is a critical need to identify new diagnostic targets for P. falciparum and develop diagnostic reagents that will specifically detect these targets with high sensitivity. Unfortunately, the identification of optimal diagnostic targets for specific pathogens has proven difficult. Most diagnostic targets have been identified by trial and error. Here, we propose to use a novel high throughput screening procedure that we have developed to identify novel diagnostic targets for P. falciparum. This procedure is based on the use of single-chain antibody (scFv) phage display and is designed to rapidly identify both novel diagnostic targets and high-affinity capture and detection antibodies specific for these targets. Advantages of this procedure include its speed, reducing the time required for the identification of both diagnostic targets and diagnostic capture/detection antibody pairs from several years to a few months, and its fidelity, specifically identifying antibody combinations that will provide the greatest clinical sensitivity when deployed in RDTs. Our proposal includes 3 Specific Aims. First, we will generate a P. falciparum-specific immune scFv phage display library using a novel phagemid vector that allows a much more rapid screening and characterization of scFv clones. Second, we will apply our novel screening procedure to this library to identify the optimal P. falciparum diagnostic targets and diagnostic antibody pairs specific to these targets. Third, we will identify those diagnostic antibody pairs that have the greatest potential clinical utility for the diagnosis of malaria in terms of sensitivity and breadth of reactivity against multiple P. falciparum isolates. The successful completion of these studies will result in the identification of novel diagnostic targets for P. falciparum and provide validated diagnostic antibodies specific for these targets, which can be used to detect infection by HRP2-deleted P. falciparum strains. In addition, this work will validate our novel screening procedure as an effective means to identify novel diagnostic targets and antibodies, which may then be applied to a wide range of infectious diseases for which accurate rapid diagnostics do not currently exist due to a lack of validated diagnostic targets.